Project 3 will focus on efforts to understand and overcome acute resistance to BRAF(V600E) suppression in melanoma by understanding how BRAF(V600E) modulates death signals. Multiple studies show that BRAF(V600E) suppression strongly induces BIM, a pro-apoptotic factor. However very little apoptosis occurs. Instead either MCL1 or BCL2A1 bind & sequester BIM. We report BCL2A1 as a newly recognized genomically amplified melanoma oncogene in about 1/3 of patients. It is also transcriptionally regulated by MITF, a lineage specific master transcription factor which is also amplified in ~20% of melanomas. BCL2A1 and MITF are thus lineage-restricted anti-apoptotic regulators. We found that BRAF-MEK-MAPK phosphorylates MITF, triggering ubiquitin-dependent degradation. Conversely BRAF(V600E) suppression blocks MITF degradation, thereby upregulating MITF. In turn MITF's targets are potently inducedsome of which are anti-apoptotic while others serve as immune antigens. The immune system may contribute to efficacy of BRAF(V600E) inhibitors because 1) treatment induces T cell tumor infiltrates within 10 days, 2) melanocytic antigen expression is strongly induced via MITF, and 3) BRAF(\/600E) targeting is significantly weaker for melanomas in an immunodeficient background. To pursue these questions Aim1 will examine MITF's and BCL2A1's roles as apoptosis antagonists to BRAF inhibitors. We will utilize clinically annotated patient specimens to determine whether genomic amplifications in MITF or BCL2A1 are prognostic for response to BRAF(V600E) targeting. Further, the use of gene targeting (knockdown) suggests that MITF and BCL2A1 are lineage specific antagonists to inhibition of BRAF sensitivity. Therefore several small molecules which antagonize MITF or BCL2A1, and which are clinically available, will be tested alone or with BRAF suppression in vitro, in mice, and (for MITF antagonism) in a recently opened clinical trial. Aim 2 will dissect interactions between BRAF(V600E), MITF, and melanoma immune responses in immune-intact mice. Inhibitors to BRAF, MEK, or ERK will be combined with immune checkpoint blocking antibodies (anti-CTLA4 anti-PDLI, or anti-PDI) and tested in mice, and in correlative studies for a human trial for vemurafenib+ipilimumab. Distinct vulnerabilities in resistant lines will be analyzed against copy number and deep sequencing data (Project 1, 2 & Cores A, B) and shRNA functional screens (Projects 1 & 2) to identify drugable combination pathways for preclinical and clinical development.